RNA Transesterification Research Articles

Oligonucleotides containing abasic threoninol-terpyridine residues as potential artificial ribonucleases

Artificial ribonucleases, also known as synthetic ribozymes, were synthesized with an internal, stereochemically-pure, abasic threoninol backbone-residue to which the RNA transesterification catalyst copper (II) terpyridine was covalently linked. These oligonucleotide conjugates were constructed to determine if the stereochemistry of the abasic threoninol backbone residue influences the transesterification rate of complementary RNA oligonucleotides. Following synthesis, these compounds were reacted with complementary 28-mer and 159-mer RNA substrates and their relative transesterification efficiencies were determined. The transesterification kinetics were also compared with previously synthesized oligonucleotides that incorporated copper (II) terpyridine via a serinol-residue. It was determined that oligonucleotides that contained copper (II) terpyridine linked via a (2S,3S)-threoninol backbone were more efficient at RNA transesterification than their (2R,3R)-stereoisomer counterpart.

A Multidimensional B-Spline Correction for Accurate Modeling Sugar Puckering in QM/MM Simulations.

The computational efficiency of approximate quantum mechanical methods allows their use for the construction of multidimensional reaction free energy profiles. It has recently been demonstrated that quantum models based on the neglect of diatomic differential overlap (NNDO) approximation have difficulty modeling deoxyribose and ribose sugar ring puckers and thus limit their predictive value in the study of RNA and DNA systems. A method has been introduced in our previous work to improve the description of the sugar puckering conformational landscape that uses a multidimensional B-spline correction map (BMAP correction) for systems involving intrinsically coupled torsion angles. This method greatly improved the adiabatic potential energy surface profiles of DNA and RNA sugar rings relative to high-level ab initio methods even for highly problematic NDDO-based models. In the present work, a BMAP correction is developed, implemented, and tested in molecular dynamics simulations using the AM1/d-PhoT semiempirical Hamiltonian for biological phosphoryl transfer reactions. Results are presented for gas-phase adiabatic potential energy surfaces of RNA transesterification model reactions and condensed-phase QM/MM free energy surfaces for nonenzymatic and RNase A-catalyzed transesterification reactions. The results show that the BMAP correction is stable, efficient, and leads to improvement in both the potential energy and free energy profiles for the reactions studied, as compared with ab initio and experimental reference data. Exploration of the effect of the size of the quantum mechanical region indicates the best agreement with experimental reaction barriers occurs when the full CpA dinucleotide substrate is treated quantum mechanically with the sugar pucker correction.

Mechanistic insights into RNA transphosphorylation from kinetic isotope effects and linear free energy relationships of model reactions.

Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate-controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition-state structure and bonding optimally requires the use of theoretical models. In this work, we apply density-functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2'-O-transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well-studied series of phosphate and phosphorothioate mono-, di- and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non-enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate-KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.

Linear free energy relationships in RNA transesterification: theoretical models to aid experimental interpretations.

RNA cleavage transesterification is of fundamental reaction in biology that is catalyzed by both protein and RNA enzymes. In this work, a series of RNA transesterification model reactions with a wide range of leaving groups are investigated with density-functional calculations in an aqueous solvation environment in order to study linear free energy relationships (LFERs) and their connection to transition state structure and bonding. Overall, results obtained from the polarizable continuum solvation model with UAKS radii produce the best linear correlations and closest overall agreement with experimental results. Reactions with a poor leaving group are predicted to proceed via a stepwise mechanism with a late transition state that is rate controlling. As leaving group becomes more acidic and labile, the barriers of both early and late transition states decrease. LFERs for each transition state are computed, with the late transition state barrier showing greater sensitivity to leaving group pKa. For sufficiently enhanced leaving groups, the reaction mechanism transits to a concerted mechanism characterized by a single early transition state. Further linear relationships were derived for bond lengths and bond orders as a function of leaving group pKa and rate constant values that can be used for prediction. This work provides important benchmark linear free energy data that allows a molecular-level characterization of the structure and bonding of the transition states for this important class of phosphoryl transfer reactions. The relations reported herein can be used to aid in the interpretation of data obtained from experimental studies of non-catalytic and catalytic mechanisms.

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.

RtcB, a Novel RNA Ligase, Can Catalyze tRNA Splicing and HAC1 mRNA Splicing in Vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

Metal-Dependent Global Folding and Activity of the 8-17 DNAzyme Studied by Fluorescence Resonance Energy Transfer

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ >> Zn2+ >>Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.

Transesterification Thio Effects of Phosphate Diesters: Free Energy Barriers and Kinetic and Equilibrium Isotope Effects from Density-Functional Theory

Primary and secondary kinetic and equilibrium isotope effects are calculated with density-functional methods for the in-line dianionic methanolysis of the native (unsubstituted) and thio-substituted cyclic phosphates. These reactions represent reverse reaction models for RNA transesterification under alkaline conditions. The effect of solvent is treated with explicit (single and double) water molecules and self-consistently with an implicit (continuum) solvation model. Singly substituted reactions at the nonbridging O(P1) position and bridging O(2)('), O(3)('), and O(5)(') positions and a doubly substituted reaction at the O(P1) and O(P2) positions were considered. Aqueous free energy barriers are calculated, and the structures and bond orders of the rate-controlling transition states are characterized. The results are consistent with available experimental data and provide useful information for the interpretation of measured isotope and thio effects used to probe mechanism in phosphoryl transfer reactions catalyzed by enzymes and ribozymes.

Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions

The glmS ribozyme is a riboswitch class that occurs in certain Gram-positive bacteria, where it resides within mRNAs encoding glucosamine 6-phosphate synthase. Members of this self-cleaving ribozyme class rapidly catalyze RNA transesterification upon binding GlcN6P, and genetic evidence suggests that this cleavage event is important for down-regulating GlmS protein expression. In this report, we present a refined secondary structure model of the glmS ribozyme and determine the importance of a conserved pseudoknot structure for optimal ribozyme function. Analyses of deletion constructs demonstrate that the pseudoknot, together with other structural elements, permits the ribozyme to achieve maximum rate constants for RNA cleavage at physiologically relevant Mg2+ concentrations. In addition, we show that substantial rate enhancements are supported by an exchange-inert cobalt (III) complex and by molar concentrations of monovalent ions. Our findings indicate that the glmS ribozyme forms a complex structure to employ catalytic strategies that do not require the direct participation of divalent metal ions.

Density Functional Study of the In-Line Mechanism of Methanolysis of Cyclic Phosphate and Thiophosphate Esters in Solution: Insight into Thio Effects in RNA Transesterification

Density functional calculations of thio effects on the in-line mechanism of methanolysis of ethylene phosphate (a reverse reaction model for RNA phosphate transesterification) are presented. A total of 12 reaction mechanisms are examined using the B3LYP functional with large basis sets, and the effects of solvation were treated using the PCM, CPCM, and SM5 solvation models. Single thio substitutions at all of the distinct phosphoryl oxygen positions (2', 3', 5', pro-R) and a double thio substitution at the nonbridging (pro-R/pro-S) positions were considered. Profiles for each reaction were calculated in the dianionic and monoanionic/monoprotic states, corresponding to reaction models under alkaline and nonalkaline conditions, respectively. These models provide insight into the mechanisms of RNA transesterification thio effects and serve as a set of high-level quantum data that can be used in the design of new semiempirical quantum models for hybrid quantum mechanical/molecular mechanical simulations and linear-scaling electronic structure calculations.

Viral RNA-directed RNA Polymerases Use Diverse Mechanisms to Promote Recombination between RNA Molecules

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.

Synthesis and Evaluation of RNA Transesterification Efficiency Using Stereospecific Serinol-Terpyridine Conjugates

Six novel artificial ribonucleases were synthesized employing a stereochemically pure abasic serinol backbone residue for attachment of the RNA transesterification agent copper(II) terpyridine. These stereochemically pure abasic residues were synthesized as phosphoramidite building blocks from the parent l -serine and d -serine starting building blocks and incorporated into oligonucleotides via solid-phase DNA synthesis. These artificial ribonucleases were constructed to determine if the stereochemistry of the alpha carbon of an abasic serinol residue has influence over RNA transesterification through selective placement of a pendant transesterification agent in either the major or minor groove. The novel artificial ribonucleases and previously synthesized artificial ribonucleases were challenged with a 28-mer and 159-mer RNA substrate. It was determined that the stereochemistry of the carbon atom derived from the α-carbon of serine did not influence the extent of cleavage in these studies using copper(II) terpyridine conjugated artificial ribonucleases.

Ribozyme speed limits.

The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2'-oxygen atom carries out an SN2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.

La(3+)-Catalyzed methanolysis of hydroxypropyl-p-nitrophenyl phosphate as a model for the RNA transesterification reaction.

The methanolysis of hydroxypropyl-p-nitrophenyl phosphate (HPNPP, 1) promoted by La(OTf)(3) under buffered conditions was studied in methanol as a function of pH at 25 degrees C. (31)P NMR studies at -90 degrees C indicate that there are at least three La/1 complexes formed at pH approximately 5.3 of 1:1, 2:2, and 1:2 stoichiometry. Kinetic studies of the observed pseudo-first-order rate constants for the methanolysis of 1 as a function of [La(3+)] at 4.5 < pH < 10.5 indicate there are two general pH regimes. In the low pH regime between 4.5 and 7.6, the plots of k(obs) versus [La(3+)] exhibit saturation behavior with very strong 1:1 binding, with a plateau rate constant that depends on [OCH(3)(-)]. The catalytically productive species is shown to be a 2:2 complex of La(3+) and 1, where the phosphate is proposed to be doubly activated, thereby promoting the methoxide reaction by some 4.6 x 10(10)-fold. In the high pH regime from 7.9 to 10.5, 1:1, 2:2, and 2:1 La(3+)/1 complexes are formed with the La(3+) coordinated in the form of [La(3+)(OCH(3)(-))](1,2). Throughout this pH regime at high [La(3+)], a saturation complex, (La(3+)OCH(3)(-))(2)/1, is formed that spontaneously decomposes with a rate constant of (5-10) x 10(-)(3) s(-)(1), leading to an acceleration of 10(9)-fold at pH 8.0.

Probing the importance of electrostatic interactions of Ce(iii) with the phosphodiester backbone during transesterification using methyl phosphonates

This paper investigates aspects of the fundamental mechanism of transesterification and hydrolysis. The ability of remote phosphates to coordinate and deliver metal catalysts to the active site (the 2′-OH) of an RNA residue is reported. Previous literature reports show that ApUp is cleaved much faster than ApU at the internal phosphodiester. This supports the accepted mechanism whereby remote phosphates coordinate and deliver metal catalysts to the active site to promote transesterification of RNA. We report that remote phosphates do not recruit metal catalysts from the bulk solution in a productive manner for transesterification. By using a polyanionic substrate, termed embedded RNA, and methyl phosphonates to mask the charge at single positions in the polyanionic backbone, we report rate increases in transesterification with methyl phosphonate substitution adjacent to the cleavage site with aqueous Ce(III) and a Ce(III) hexaaza macrocycle.

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by CuII Terpyridine

The hydrolysis and transesterification of RNA are catalyzed by a variety of metal ions, metal complexes, metalloenzymes, and ribozymes. These reactions are of fundamental biochemical importance. However, the role that metal ions play in RNA hydrolysis is not completely understood. We previously showed that aqueous Cu(II) terpyridine (Cutrpy) is effective for both transesterification and hydrolysis of RNA, and we harnessed this reactivity by constructing ribozyme mimics that employ terpyridyl Cu(II) in their active sites. Here we report a detailed kinetic study of the hydrolysis of adenosine-2‘,3‘-cyclic monophosphate (cAMP) by Cutrpy. The reaction is established to be first-order in Cutrpy and first-order in substrate. Catalytic turnover is observed, although product inhibition occurs. Chloride ion also inhibits the reaction, which indicates the disadvantage of using NaCl as an ionic strength buffer in related studies. The pH−rate profile is sigmoidal and implicates the hydroxide form of the catalyst, CutrpyOH+, as the active species. Isotope effects were used to determine whether the metal hydroxide acts as a nucleophile or a base. The solvent deuterium kinetic isotope effect, kH/kD, was measured to be 1.0 ± 0.1 after considering equilibrium isotope effects. To assist the mechanistic interpretation of the measured isotope effects, the fractionation factors for a dianionic phosphorane transition state (methyl phosphate dianion) and hydroxide were evaluated by ab initio quantum chemical calculations. Considering the calculated results along with the relevant experimental fractionation factors, isotope effects were predicted for three overall mechanisms: nucleophilic catalysis, general base catalysis, and specific base/general acid catalysis. We conclude that CutrpyOH+ acts as a nucleophilic catalyst in the hydrolysis of cAMP. This contrasts with the behavior of CutrpyOH+ as a base catalyst in the transesterification of RNA.

Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2‘-Hydroxyl Group

A detailed understanding of the susceptibility of RNA phosphodiesters to specific base-catalyzed cleavage is necessary to approximate the stability of RNA under various conditions. In addition, quantifying the rate enhancements that can be produced exclusively by this common cleavage mechanism is needed to fully interpret the mechanisms employed by ribonucleases and by RNA-cleaving ribozymes. Chimeric DNA/RNA oligonucleotides were used to examine the rates of hydroxide-dependent degradation of RNA phosphodiesters under reaction conditions that simulate those of biological systems. Under neutral or alkaline pH conditions, the dominant pathway for RNA degradation is an internal phosphoester transfer reaction that is promoted by specific base catalysis. As expected, increasing the concentration of hydroxide ion, increasing the concentration of divalent magnesium, or raising the temperature accelerates strand scission. In most instances, the identities of the nucleotide bases that flank the target RNA linkage have a negligible effect on the pKa of the nucleophilic 2‘-hydroxyl group, and only have a minor effect on the maximum rate constant for the transesterification reaction. Under representative physiological conditions, specific base catalysis of RNA cleavage generates a maximum rate enhancement of ∼100 000-fold over the background rate of RNA transesterification. The kinetic parameters reported herein provide theoretical limits for the stability of RNA polymers and for the proficiency of RNA-cleaving enzymes and enzyme mimics that exclusively employ a mechanism of general base catalysis.

The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction

The hairpin ribozyme is an example of a small catalytic RNA that catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. We have utilised chemical synthesis of RNA to create mutants of the hairpin ribozyme in which a nucleoside analogue replaces one of the essential pyrimidines in the ribozyme. Individual pyrimidine nucleosides were substituted by 4-thiouridine, O 4-methyluridine, O 2-methyluridine or 2-pyrimidinone-1-β- d-riboside. To facilitate the synthesis of oligoribonucleotides containing 4-thiouridine, we have devised a new synthetic route to the key intermediate 5′- O -(4,4′-dimethoxytrityl)-2′- O- tert-butyldimethylsilyl- S-cyanoethyl-4-thiouridine. The ability of the modified ribozymes to support catalysis was studied and the steady-state kinetic parameters were determined for each mutant. The range of analogues used in this study allows the important functional groups of the essential pyrimidines to be identified. The results demonstrate that each pyrimidine (U 41, U 42 and C 25) plays an important role in hairpin ribozyme catalysis. The findings are discussed in terms of the various models that have been proposed for loop B of the hairpin ribozyme.

Transesterification of RNA by Cu(II) terpyridine

We have used an RNA/DNA chimera, reffered to as embRNA, to study the transesterification of RNA catalyzed by aqueous terpyridyl Cu(II). This substrate is clevaed in only one position, but it contains the polyanionic charge and many of the metal binding sites of an RNA polymer. The pH rate profile and substrate dependence of the reaction are reported. Kinetic results are consistent with the active agent being the monohydroxy form of the Cu(II) complex.

Metal ions play a passive role in the hairpin ribozyme catalysed reaction.

The hairpin ribozyme is an example of a small catalytic RNA which catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. The hairpin ribozyme, in common with all other small ribozymes such as the hammerhead, requires the presence of a divalent metal ion co-factor (typically magnesium) for the reaction to take place. To investigate the role of magnesium ions in the hairpin catalysed reaction we have synthesised two epimeric modified substrates in which a phosphorothioate replaces the scissile phosphodiester bond. Previously, Burke and co-workers have reported that no thio-effect is observed with the Rp-phosphorothioate isomer. We observe the absence of a thio-effect with both diastereomeric phosphorothioate hairpin substrates. Furthermore we report that inert cobalt (III) complexes are capable of supporting the hairpin ribozyme reaction, with a similar efficiency to Mg2+,even in the presence of EDTA. Variation of the net charge on the inert cobalt complex does not change the observed rate of reaction. These results suggest that metal ions play a passive role in the hairpin ribozyme catalysed reaction and are probably required for structural purposes only. This places the hairpin ribozyme in a different mechanistic class to other small ribozymes such as the hammerhead.